Project Summary We will use an integrative and multidisciplinary approach to investigate how the S1 head domain of the myosin heavy chain (MHC) protein drives muscle function. Myosin is the molecular motor of muscle and the major component of myofibrillar thick filaments. Its ATP-dependent interaction with actin-containing thin filaments powers muscle contraction. Our studies use the model organism Drosophila melanogaster, which has a single muscle Mhc gene but produces multiple forms of the protein (isoforms) by alternative RNA splicing. Using MHC null mutants in conjunction with germline transformation, we express engineered versions of the protein and employ them to test basic and novel hypotheses that predict structural, biochemical, fiber mechanical, physiological and locomotory properties imparted by specific myosin domains and amino acid residues. An innovative aspect of our system is that functions will be tested in vitro, in skeletal and cardiac muscle and in intact organisms. Therefore, we can determine directly and to what degree a specific biochemical property defines a physiological or locomotory characteristic. To this end, we will utilize a battery of in vitro and in vivo assays: ATPase, actin and nucleotide affinity, in vitro motility, x-ray crystallography, molecular modeling, electron microscopy, isolated fiber mechanics, video-based cardiac imaging and organismal locomotion. Our first aim is to elucidate the role of a critical communication element of the myosin motor called the relay domain. We will determine the importance of specific transient interactions of key amino acid residues of the relay that we hypothesize to interact with the converter domain or with the SH1-SH2 helix region during the mechanochemical cycle. For this, we will combine the transgenic approach with classical genetics to introduce and suppress mutations. Our second aim will test predicted isoform-specific interactions during the mechanochemical cycle. To this end, we will exploit the Drosophila system to express flight and embryonic muscle myosin isoforms that will be crystallized and compared in multiple nucleotide binding states. This approach will also be used for structural analysis of human beta-cardiac myosin, which will be the first mammalian striated muscle myosin analyzed at atomic resolution. Our third aim will test our hypotheses about the effects of a mutation in myosin that is known to cause restrictive cardiomyopathy. We will create a Drosophila model of this human disease by mutating the invariant proline at the myosin head-rod junction. We will define the biochemical, biophysical, mechanical and locomotory defects engendered by the myosin mutation. We will also examine whether the mutation affects the flexibility of the myosin head and determine how it influences Drosophila heart (dorsal vessel) structure and function. Overall, our novel integrative analyses will permit testing of models for the transduction of chemical energy into movement and will yield insight into how myosin functions in muscle. Further, we will directly address the role of myosin in human muscle disease, by defining the molecular basis of a restrictive cardiomyopathy.